The stimulative effect of glucocorticoids on the maintenance of fluid and electrolyte balance has been known for more than two decades. Pharmacological doses of methylprednisolone stimulated Na+ absorption in small animals. However, molecular mechanisms underlying this activation remain elusive. Na+/H+ exchanger NHE3 in the brush border membrane of intestine and kidney plays a major role in transepithelial Na+ absorption. We previously demonstrated that methylprednisolone in rabbits specifically stimulated NHE3 mRNA in ileum without affecting NHE1 mRNA levels. Others have shown that glucocorticoids specifically activated NHE3 mRNA in ileum, proximal colon and renal proximal tubules. These results suggest that glucocorticoids activate NHE3 activity by gene expression of NHE3. However, we recently found using Caco-2 cells that dexamethasone activates NHE3 transport without affecting NHE3 mRNA expression. This non-transcriptional activation of NHE3 by dexamethasone was demonstrated in OK cells, suggesting that transcriptional activation of NHE3 may not be the only determining factor in glucocorticoid-stimulation of NHE3. We found that dexamethasone enhanced NHE3 activity only in the presence of a NHE3 regulatory protein, NHERF2. We identified serum- and glucocorticoids-induced protein kinase 1, SGK1, as a protein interacting with PDZ domains of NHERF2. We demonstrated that SGK1 activated NHE3 activity and expression of "kinase-dead" SGK1 in OK cells markedly blocked the dexamethasone effect, demonstrating the importance of SGKI. We also showed that SGK1 directly phosphorylated NHE3 in vitro suggesting phosphorylation-dependent regulation of NHE3. In this application, we propose to investigate: (1) the roles of SGK1 and NHERF2 in activation of NHE3 by glucocorticoids; (2) molecular mechanisms underlying the activation of NHE3 by SGK1 and NHERF2 in response to glucocorticoid; and (3) the interaction between SGK1, NHERF2 and NHE3. This proposal will resolve complexity in NHE3 regulation by glucocorticoids, and enhance our understanding of protein-protein interaction in regulation of membrane transport proteins. [unreadable] [unreadable]